The overall direction of the Molecular Mechanisms of Tumor Promotion Section is to understand the regulation of the signaling pathways downstream from the lipophilic second messenger diacylglycerol, to elucidate the basis for heterogeneity of response to different ligands which function through this pathway, and to exploit this understanding for developing novel ligands with unique behavior that function through this pathway. A complementary direction is to understand the regulation and structure activity relations for the vanilloid receptor. The vanilloid receptor is a downstream target of the diacylglycerol signaling pathway, shares partial homology in its ligands to this pathway, and shares with the diacylglycerol signaling pathway an important role in inflammation. Both directions impact both our understanding of biological regulation and the potential development of therapeutic agents. Protein kinase C, the best studied downstream target for diacylglycerol, represents the classic system for tumor promotion and is a therapeutic target for cancer chemotherapy. The vanilloid receptor represents a promising therapeutic target for cancer pain, among other indications, and thus represents an important direction in palliative care for cancer patients. DAG-lactones represent a synthetically accessible platform for probing the structure activity relations of protein kinase C and the other targets downstream of the second messenger diacylglycerol. We have continued to evaluate DAG-lactones designed to have selectivity for the RasGRP subclass of diacylglycerol targets. This class is of particular importance because it functions as an activator of Ras, which is many tumors shows enhanced activity without being mutated. Selectivity is being evaluated both at the in vitro level and in intact cells. Bryostatin is an agent in clinical trials with a unique mechanism of action. It binds to protein kinase C with high affinity and activates the enzyme but paradoxically antagonizes many protein kinase C mediated responses. We find, using microarray analysis of gene expression and aptamer arrays for analysis of protein expression, along with detailed examination of the time and dose dependence of genes representative of the differences in expression revealed by the microarray analysis, that transient duration is the predominant difference in the mode of action of bryostatin as compared to typical protein kinase C activators such as the phorbol esters. In collaboration with the groups of Gary Keck and Michael Krische, we seek to define the critical structural elements in bryostatin conferring its unique pattern of activity, with the goal of developing the next generation of bryostatin analogues. An important recent advance was to show that the upper portion of the bryostatin molecule is not only necessary but also sufficient for conferring the unique pattern of biological response to the bryostatin. In other studies, we have begun to analyze hybrid ligands that contain an upper region with similarities to that of the upper portion of bryostatin together with a bottom half comprising a DAG-lactone to confer binding acitivity. Protein kinase C is subject to post-translational modification, in particular phosphorylation. In collaboration with the CCR Collaborative Protein Technology Resource, we have shown that the extent of protein kinase C modification is much more extensive than had been recognized. Moreover, the pattern of modification was different for different ligands such as phorbol ester or bryostatin. We suggest that such modification signatures may be of particular value for the structure activity evaluation of ligands with complex effects where, as in the case of protein kinase C, ligand binding causes both activation and change in subcellular localization. Comparing the various cell lines of the NCI60 cell line panel, we have shown that the patterns of PKC delta modification show marked differences between the various cell lines. Modification of DAG signaling in these various cell lines causes changes in the patterns of modification, but the changes are much smaller than those associated with phorbol ester, emphasizing that phorbol ester treatment in not a good surrogate for physiological levels of DAG. In addition to those proteins that recognize DAG and phorbol esters through their C1 domains, other proteins contain homologous C1 domains that fail to recognize DAG or phorbol ester. We are actively involved in understanding the structural basis for these differences. Systems examined include the C1 domains of RasGRP2 and Vav2/3 as well as the C1a domain of PKC theta. On our TRPV1 project, we have made considerable progress in defining the optimal structures for binding of antagonists to human TRPV1, as part of our effort which seeks to advance the development of drug candidates for this target. We are exploring the use of in silico screening to define new classes of ligands for TRPV1 and for TRPV2. In analogy with our studies on C1 domains, we are seeking to extend our understanding of ligand binding to TRPV1 to other family members such as TRPV2.